Transcript-based Library Construction Based on Tn5 Transposase Develops A New Era of Rapid Library
Posted: Jul 06, 2020
In the routine construction of transcriptomes, RNA needs to be converted into double-stranded DNA first, and it needs to go through multiple steps of target RNA enrichment and fragmentation, first and second strand cDNA synthesis, linker connection, PCR amplification, etc. The operation is cumbersome and time-consuming, and the synthesis of double-stranded cDNA requires the introduction of an additional DNA polymerase, which may result in an increased preference for sequencing. Therefore, for the majority of scientific researchers, it is extremely urgent to explore new technologies for rapid and efficient database construction.
Recently, scientists have developed a new rapid library building method based on Tn5 transposase transcriptome sequencing, named after the name of red wine - SHERRY (Sequencing HEteRo RNA-DNA-hYbrid). Compared with the existing transcriptome sequencing, cDNA two-strand synthesis is not required, which greatly simplifies the process of database construction; and only requires a small amount of samples, and can be used for high-quality single-cell transcriptome sequencing.
Since the popularity of NGS sequencers and the rapid decline in sequencing costs, transcriptome analysis by RNA-seq has become a routine method in biomedical research. RNA-seq has been widely used to solve various biological problems, from exploring the pathogenesis of diseases to constructing transcriptome maps of various species. For different studies, different starting amounts are required, and different amplification methods are used. The operation is complicated and time-consuming, which is not conducive to experiments with multiple samples.
The bacterial Tn5 transposase has been used in next-generation sequencing, using the unique "tag" function of the dimeric Tn5, which can cut double-stranded DNA (dsDNA) and connect the resulting DNA ends to specific connectors. The genetically engineered Tn5 is widely used in the preparation of sequencing libraries due to its rapid synthesis ability and low sample input. For general library preparation, Tn5 directly reacts with dsDNA, which can break double-stranded DNA in one step and make the two ends of the DNA carry a linker. This simple one-step tagging reaction greatly simplifies the experimental process, shortens the working time and reduces the cost, and is widely used in DNA sequencing library construction technology.
As a member of the RNase H superfamily, Tn5 binds to RNA/DNA heteroduplex similarly to dsDNA and efficiently fragments, and then connects specific amplification and sequencing adapters to the hybrid. This method is called SHERRY, which can greatly simplify the experimental steps and reduce the input. SHERRY can handle a variety of input samples from single cells to large amounts of RNA, and its dynamic range spans six orders of magnitude. Compared with other commonly used methods, SHERRY shows excellent cross-sample stability and comparability for large-molecule RNA and single cells, and provides a unique solution for small batch samples that are difficult to handle with existing methods.
SHERRY consists of three parts: RNA reverse transcription, RNA/cDNA hybrid chain labeling and PCR amplification. SHERRY method transcriptome rapid library construction process: after reverse transcription of different types of RNA through oligo-dT primers, Tn5 transposase is randomly combined on the RNA/DNA hybrid strand for cleavage. After tagmentation, the transposase will add adaptor adaptors at both ends of the hybrid strand, followed by an extension reaction to obtain complete double-stranded DNA containing adaptors, and finally the PCR library amplification will be performed under the action of sequencing primers.
After PCR amplification, no matter whether RNase H digestion or not, high purity products were obtained. Both reaction test conditions and SHERRY sequencing results showed>90% human genome localization rate (~80% exon rate) and Nearly 12,000 genes were detected, which verified the source of the library's transcriptome. In summary, it was confirmed that the Tn5 transposase can label the DNA and RNA strands of RNA/DNA heteroduplexes.
The scientists also investigated whether SHERRY can construct RNA-seq libraries from single cells. First, reduce the input to 100 pg total RNA, which is equivalent to about 10 cells of RNA. SHERRY's results are of high quality, with high localization and exon incidence, and detected nearly 9,000 genes. The scSHERRY method shows higher sequencing quality and lower preference in single cell transcriptome sequencing. Overall, compared to Smart-seq2, scSHERRY produced libraries of comparable quality and lower GC bias.
Based on the fragmentation principle of Tn5 transposase directly acting on the RNA/DNA hybrid strand, a new method for rapid library construction of transcriptome sequencing was developed-SHERRY, which further expanded the new uses of Tn5 transposase and not only solved the problems of the cumbersome, time-consuming and large preference of the conventional RNA-seq process, but also greatly reduces the cost. Therefore, compared with the traditional RNA library preparation method, the SHERRY method for rapid library construction has greater competitiveness in drug development.
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